per
WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau
INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(51) International Patent Classification 5 :
A61K 39/395, C07K 15/28
Al
(11) International Publication Number:
WO 91/16928
(43) International Publication Date : 1 4 November 1991 (14.11.91)
(21) International Application Number : PCT/US9 1 /02946
(22) International Filing Date: 29 April 1991 (29.04.91)
(30) Priority data:
9009548.0
27 April 1990 (27.04.90)
GB
(71) Applicants (for all designated States except US): CELL-
TECH LIMITED [GB/GB]; 216 Bath Road, Slough,
Berkshire SL1 4EN (GB).
(71) Applicant (for all designated States except US): BO EH-
RINGER INGELHEIM PHARMACEUTICALS, INC.
[US/US]; 90 East Ridge, P.O. Box 368, Ridgefield, CT
06877 (US).
(72) Inventors ; and
(75) Inventors/Applicants (for US only) : ADAIR, John, Robert
[GB/GB] ; 23 George Road, Stokenchurch, High Wy-
combe, Buckinghamshire HP14 3RN (GB). ROBIN-
SON, Martyn, Kim [GB/GB]; 62 Strawberry Vale,
Twickenham, Middlesex TW1 4SE (GB). BRIGHT, Sus-
an, Margaret [GB/GB] ; 24 Pound Lane, Marlow, Buck-
inghamshire SL7 2AY (GB). ROTHLEIN, Robert, A.
[US/US]; 32 Tamanny Trail, Danbury, CT 06811 (US).
(74) Agents : FOX, Samuel, L. et al. ; Sterne, Kessler, Goldstein
& Fox, 1225 Connecticut Avenue, N.W., Suite 300,
Washington, DC 20036 (US).
(81) Designated States: AT, AT (European patent), AU, BB, BE
(European patent), BF (OAPI patent), BG, BJ (OAPI
patent), BR, CA, CF (OAPI patent), CG (OAPI patent),
CH, CH (European patent), CM (OAPI patent), DE,
DE (European patent), DK, DK (European patent), ES,
ES (European patent), FI, FR (European patent), GA
(OAPI patent), GB, GB (European patent), GR (Euro-
pean patent), HU, IT (European patent), JP, KP, KR,
LK, LU, LU (European patent), MC, MG, ML (OAPI
patent), MR (OAPI patent), MW, NL, NL (European
patent), NO, PL, RO, SD, SE, SE (European patent),
SN (OAPI patent), SU, TD (OAPI patent), TG (OAPI
patent), US.
Published
With international search report.
(54) Title: HUMANIZED CHIMERIC ANTI-ICAM-1 ANTIBODIES, METHODS OF PREPARATION AND USE
(57) Abstract
The present invention discloses humanized chimeric antibodies which are capable of binding to the intercellular adhesion
molecule ICAM-1. Specifically, disclosed are humanized anti-ICAM-1 antibodies of the IgGl, IgG2, and IgG4 subtype. These
antibodies are useful in treating specific and non-specific inflammation, rhinoviral infection, HIV infection, the dissemination of
HIV infected cells, and asthma. In addition, the humanized antibodies disclosed can be useful in methods of diagnosing and lo-
calizing sites of inflammation and infection and tumors expressing ICAM-1.
FOR THE PURPOSES OF INFORMATION ONLY
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.
AT
Austria
ES
Spain
MG
Madagascar
AU
Australia
Fl
Finland
ML
Mali
BB
Barbados
FR
France
MN
Mongolia
BE
Belgium
GA
Gabon
MR
Mauritania
BF
Burkina Faso
GB
United Kingdom
MW
Malawi
BG
Bulgaria
GN
Guinea
NL
Netherlands
BJ
Benin
GR
Greece
NO
Norway
BR
Brazil
HU
Hungary
PL
Poland
CA
Canada
IT
Italy
RO
Romania
CF
Central African Republic
JP
Japan
SD
Sudan
CG
Congo
KP
Democratic People's Republic
SE
Sweden
CH
Switzerland
of Korea
SN
Senegal
CI
Cote dMvoiru
KR
Republic of Korea
Liechtenstein
su
Soviet Union
CM
Cameroon
LI
TD
Chad
CS
Czechoslovakia
LK
Sri Lanka
TG
Togo
DE
Germany
LU
Luxembourg
US
United Slates of America
DK
Denmark
MC
Monaco
WO 91/16928 PCT/US91/02946
-1-
HUMANIZED CHIMERIC ANTI-ICAM-1 ANTIBODIES. METHODS
OF PREPARATION AND USE
Field of the Invention:
The present invention relates to a chimeric antibody molecule, and
5 especially a humanized chimeric antibody molecule, having specificity for an
antigenic determinant of Intercellular Adhesion Molecule 1 (ICAM-1), to a
process for its production using recombinant DNA technology and to its
therapeutic use.
In the present application, the term "chimeric antibody molecule H is
10 used to describe an antibody molecule having heavy and/or light chains
comprising at least the variable regions of heavy and/or light chains derived
for one immunoglobulin molecule linked to at least part of a second protein.
The second protein may comprise additional antibody constant regions
domains derived from a different immunoglobulin molecule or a non-
15 immunoglobulin protein. The term "humanized chimeric antibody molecule"
is used to describe a molecule having heavy and light chain variable region
domains derived from an immunoglobulin from a non-human species, the
remaining immunoglobulin constant region domains of the molecule being
derived from a human immunoglobulin. The abbreviation "MAb" is used to
20 indicate a monoclonal antibody.
The present invention also relates to the use of chimeric antibodies
capable of binding to ICAM-1 to inhibit intercellular adhesion of cells of
granulocyte or macrophage lineage. The use of such molecules provides a
method for the treatment of specific and non-specific inflammation.
WO 91/16928
PCT/US91/02946
The present invention also relates to chimeric antibody capable of
binding ICAM-1 in the treatment of viral, and particularly rhinoviral disease.
The invention also relates to therapeutic and prophylactic methods for
suppressing the infection of leukocytes with HIV, and particularly with HIV-1,
5 in an individual who is exposed to HTV or effected by HIV, and is thus in
need of such suppression through the administration of a chimeric antibody
capable of binding ICAM-1 . It therefore provides a therapy for diseases,
such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by
the HIV virus.
10 The invention also relates to a therapeutic method for suppressing the
migration of HIV-1 infected cells from the circulatory system using chimeric
antibodies capable of binding ICAM-1 . It therefore provides a therapy for
diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are
caused by the HIV-1 virus.
15 The present invention relates to the use chimeric antibodies capable of
binding ICAM-1 in the treatment of asthma.
Background of the Invention
A. Humanized antibodies
Natural immunoglobulins have been known for many years, as have
20 the various fragments thereof, such as the Fab, (Fab') 2 and Fc fragments,
which can be derived by enzymatic cleavage. Natural immunoglobulins
comprise a generally Y-shaped molecule having an antigen-binding site
towards the free end of each upper arm. The remainder of the structure, and
particularly the stem of the Y, mediates the effector functions associated with
25 immunoglobulins.
Natural immunoglobulins have been used in assay, diagnosis and, to
a more limited extent, therapy. However, such uses, especially in therapy,
have been hindered by the polyclonal nature of natural immunoglobulins. A
significant step towards the realization of the potential of immunoglobulins as
30 therapeutic agents was the discovery of techniques for the preparation of
monoclonal antibodies of defined specificity (Kohler et al., Nature 265:295-
WO 91/16928
PCI7US91/02946
497 (1975)). However, most MAbs are produced by fusions of rodent spleen
cells with rodent myeloma cells. They are therefore essentially rodent
proteins. There are very few reports of the production of human MAbs.
Since most available MAbs are of rodent origin, they are naturally
5 antigenic in humans and thus can give rise to an undesirable immune response
termed the KAMA (Human Anti-Mouse Antibody) response. Therefore, the
use of rodent MAbs as therapeutic agents in humans is inherently limited by
the fact that the human subject will mount an immunological response to the
MAb and will either remove it entirely or at least reduce its effectiveness. In
10 practice MAbs of rodent origin may not be used in a patient for more than one
or a few treatments as a HAMA response soon develops rendering the MAb
ineffective as well as giving rise to undesirable reactions.
Proposals have therefore been made for making non-human MAbs less
antigenic in humans. Such techniques can be generically termed
15 "humanization" techniques. These techniques generally involve the use of
recombinant DNA technology to manipulate DNA sequences encoding the
polypeptide chains of the antibody molecule.
In particular one procedure which has bee proposed for the preparation
of humanized antibodies is the so-called chimerization procedures.
20 Such chimerization procedures involve production of chimeric
antibodies in which an antigen binding site comprising the complete variable
domains of one antibody is linked to constant domains derived from another
antibody. Some early methods for carrying out such a chimerization
procedure are described in EP-A-0120694 (Celltech Limited), EP-A-0125023
25 (Genentech Inc. and City of Hope), EP-A-01714906 (Res. Dev. Corp. Japan),
EP-A-0173494 (Stanford University), EP-A-0194276 (Celltech Limited). The
latter Celltech application also shows the production of an antibody molecule
comprising the variable domains of a mouse MAb, the CHI and CL domains
of a human immunoglobulin, and* a non-immunoglobulin derived protein in
30 place of the Fc portion of the human immunoglobulin.
B. Leukocyte Attachment and Functions
Leukocytes and granulocytes must be able to adhere to cellular
substrates in order for an inflammatory response to occur and to properly
defend the host against foreign invaders such as viruses, bacteria, and
allergens. This fact has become evident from two converging lines of
research.
The first line of research involves studies of leukocyte membrane
proteins (Wallis, W.L, et al, J. Immunol 755:2323-2330 (1985); Mentzer,
SJ., et al, J. Cell Physiol 126:285-290 (1986); Haskard, D.O., et al, J.
Immunol 757:2901-2906 (1986); Harlan, J.M., et al, Blood 55:167-178
(1985)). Of particular importance to the process of cellular adhesion is a
family of leukocyte membrane proteins known as the "CD18 H family or
complex. This family consists of three heterodimers (known as "Mac-l,"
"LFA-1," and H P150,90 M ), all of which share a common subunit (known as
the B subunit) and a unique subunit (known as the a subunit) (Springer, T. A. ,
et al, Immunol Rev. 58:111-135 (1982); Springer, T., et al, Fed. Proc.
44:2660-2663 (1985); Keizer, G., et al, Eur. J. Immunol 75:1142-1147
(1985); Sanchez-Madrid, F., etal, J. Exper. Med. 758:1785-1803 (1983)).
Monoclonal antibodies against the CD18 family of leukocyte membrane
proteins, by acting as antagonists of these proteins, inhibit a multitude of
leukocyte adhesion dependent events in vitro . This includes the ability of
granulocytes to aggregate in response to appropriate stimuli, the ability of
granulocytes to attach to protein coated plastic, the ability of granulocytes to
migrate in 2-dimensional agarose assays, and the ability of granulocytes to
attach to endothelial cells.
The second line of research results from studies involving individuals,
who, due to an inherited flaw in the gene encoding for the common subunit
of the CD 18 family of leukocyte- adhesion molecules, are unable to express
any of these adhesion molecules on the surfaces of their cells. Such in-
dividuals are said to suffer from "leukocyte adherence deficiency disease 11
WO 91/16928
PCT/US9 1/02946
-5-
( M LAD M ) (Anderson, D.C., et al, Fed. Proc. 44:2611-2611 (1985);
Anderson, D.C., et aL, J. Infect. Dis. 752:668-689 (1985)). Characteristic
features of LAD patients include necrotic soft tissue lesions, impaired pus
formation and wound healing, as well as abnormalities of adhesion-dependent
5 leukocyte functions in vitro , and susceptibility to chronic and recurring
bacterial infections. Granulocytes from these LAD patients behave in the
same defective manner in vitro as do their normal counterparts in the presence
of anti-CD18 monoclonal antibody. That is, they are unable to perform
adhesion related functions such as aggregation or attachment to endothelial
10 cells. More importantly, however, is the observation that these patients are
unable to mount a normal inflammatory response because of the inability of
their granulocytes to attach to cellular substrates. Most remarkable is the
observation that granulocytes from these LAD patients are unable to get to
sites of inflammation such as skin infections due to their inability to attach to
15 the endothelial cells in the blood vessels near the inflammation lesions. Such
attachment is a necessary step for extravasation.
Thus, in summary, the ability of lymphocytes and granulocytes to
maintain the health and viability of an animal requires that they be capable of
adhering to other cells (such as endothelial cells). Granulocyte-endothelial cell
20 adherence has been found to require cell-cell contacts which involve specific
receptor molecules present on the granulocyte cell surface. These receptors
enable the leukocyte to adhere to other leukocytes or to endothelial, and other
non-vascular cells.
The cell surface receptor molecules of leukocytes have been found to
25 be highly related to one another. Humans whose leukocytes lack these cell
surface receptor molecules exhibit chronic and recurring infections, as well as
other clinical symptoms. Inflammation reactions are mitigated when
leukocytes are unable to adhere in a normal fashion due to the lack of
functional adhesion molecules of- the CD 18 complex. Because leukocyte
30 adhesion is involved in the process through which tissue inflammation arises,
WO 91/16928
PCT/US91/02946
-6-
an understanding of the process of leukocyte adhesion is of significant value
in defining a treatment for specific and non-specific inflammation.
Additionally, since lymphocyte adhesion is involved in the process
through which foreign body or tissue is identified and rejected, an
5 understanding of this process is of significant value in the fields of organ
transplantation, tissue grafting, allergy and oncology,
C. The Intercellular Adhesion Molecule ICAM-1 and Cellular
Adhesion
The intercellular adhesion molecule ICAM-1 was first identified and
10 partially characterized according to the procedure of Rothlein, R. et al. (J.
Immunol. 757:1270-1274 (1986)), which reference is herein incorporated by
reference. ICAM-1, its preparation, purification, and characteristics are
disclosed in WO 90/03400 which application is herein incorporated by
reference in its entirety.
15 ICAM-1 was initially realized as being involved in the process of
cellular adhesion between endothelial cells and leukocytes. Cellular adhesion
is the process through which leukocytes attach to cellular substrates, such as
endothelial cells, in order to migrate from circulation to sites of ongoing
inflammation, and properly defend the host against foreign invaders such as
20 bacteria or viruses. An excellent review of the defense system is provided by
Eisen, H.W., (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, PA
(1980), pp. 290-295 and 381-418).
One of the molecules on the surface of endothelial cells which
participates in the adhesion process is ICAM-1. This molecule has been
25 shown to mediate adhesion by binding to molecules of the CD-I 8, CD- 11/ 18
family of glycoproteins which are present on the cell surfaces of leukocytes
(Sanchez-Madrid, F. et al., J. Exper. Med. 258:1785-1803 (1983); Keizer,
G.D. etal, Eur. J. Immunol. 15:1142-1147 (1985)).
WO 91/16928
PCT/US91/02946
Intercellular Adhesion Molecule (ICAM-1) is an inducible cell surface
glycoprotein expressed on various cell types including vascular endothelial
cells, and is expressed preferentially at sites of inflammation. Since ICAM-1
is the natural binding ligand of LFA-1, ICAM-l-LFA-1 interactions play a
5 central role in cellular adhesion, recruitment of lymphocytes to sites of
inflammation and the triggering of lymphocyte functions which contribute to
both specific and non-specific inflammation.
D. The Cellular Receptor for Human Rhinovirus
Abraham et al (J. Virol 57:340-345 (1984)) discovered that the
10 majority of randomly selected human rhinovirus ( H HRV M ) serotypes were able
to bind to the same cellular receptor. A monoclonal antibody was
subsequently developed by Colonno et al. (Colonno et al. , J. Cell. Biochem.
Suppl. 10 (part D):266 (1986); Colonno et al., J. Virol. 57:7-12 (1986);
Colonno et al, European Patent Application Publication No. 169,146) which
15 was capable of blocking attachment of HRV of the major serotype to the
surfaces of endothelial cells. The endothelial cell receptor protein recognized
by this antibody was isolated and found to be a 90 kd protein (Tomassini et
al, J. Virol. 58:290-295 (1986) and later shown to be the ICAM-1 molecule
(Staunton et al, Cell 55:849-854 (1989)).
20 Treatment of rhinoviral infection, especially infection by the major
type human rhinovirus has been proposed using a murine monoclonal antibody
directed against the viral receptor, ICAM-1 (EP 391088).
E. Infection with HIV
HIV infection is the cause of AIDS. Two major variants of HTV have
25 been described: HTV-1 and HTV-2. HIV-1 is prevalent in North America and
Europe, in contrast to HTV-2 which is prevalent only in Africa. The viruses
have similar structures and encode proteins having similar function. The
nucleotide and protein sequences of the genes and gene products of the two
variants have been found to have about 40% homology with one another.
HIV infection is believed to occur via the binding of a viral protein
(termed M gpl20") to a receptor molecule (termed M CD4") present on the
surface of T4 (T helper") lymphocytes (Schnittman, S. M. et al., J.
Immunol. 747:4181-4186 (1988), which reference is incorporated herein by
reference). The virus then enters the cell and proceeds to replicate, in a
process which ultimately results in the death of the T cell. The destruction
of an individual's T4 population is a direct result of HIV infection. HIV can
be recovered from peripheral blood mononuclear cells and human plasma (/.
Clin. Microbiol. 25:2371-2376 (1988); N. Engl J. Med. 321: 1621-1625
(1989)). Results suggest more viremia than had been previously estimated and
a T-cell infection frequency as high as 1 % .
The destruction of the T cells results in an impairment in the ability of
the infected patient to combat opportunistic infections. Although individuals
afflicted with AIDS often develop cancers, the relationship between these
cancers and HIV infection is, in most cases, uncertain.
Although the mere replication of the HIV virus is lethal to infected
cells, such replication is typically detected in only a small fraction of the T4
cells of an infected individual. Several lines of research have elucidated other
mechanisms through which the HIV virus mediates the destruction of the T4
population.
Apart from through HIV replication, HIV infected cells can be
destroyed through the action of cytotoxic, killer cells. Killer cells are
normally present in humans, and serve to monitor the host and destroy any
foreign cells (such as in mismatched blood transfusions or organ transplants,
etc.) which may be encountered. Upon infection with HIV, T4 cells display
the gpl20 molecule on their cell surfaces. Killer cells recognize such T4 cells
as foreign (rather than native cells), and accordingly, mediate their
destruction.
HIV infection can also lead to the destruction of non-infected healthy
cells. Infected cells can secrete the gpl20 protein into the blood system. The
free gpl20 molecules can then bind to the CD4 receptors of healthy,
WO 91/16928 PCI7US91/02946
-9-
uninfected cells. Such binding causes the cells to take on the appearance of
HIV infected cells. Cytotoxic, killer cells recognize the gpl20 bound to the
uninfected T4 cells, conclude that the cell is foreign, and mediate the
destruction of the T4 cells.
5 An additional mechanism, and one of special interest to the present
invention, with which HIV can cause T4 death is through the formation of
"syncytia." A "syncytium" is a multinucleated giant cell, formed from the
fusion of as many as several hundred T4 cells. Infection with HIV causes the
infected cell to become able to fuse with other T4 cells. Such fusion partners
10 may themselves be HTV infected, or they may be uninfected healthy cells.
The syncytium cannot function and soon dies. Its death accomplishes the
destruction of both HTV infected and HIV uninfected T4 cells. This process
is of special interest to the present invention since it entails the direct cell-cell
contact of T4 cells. The ability of HIV-infected cells to form syncytia
15 indicates that such cells acquire a means for fusing with healthy cells. Thus,
cell-cell contacts may be of fundamental importance in the process through
which HTV infection is transmitted from one cell to another within an
individual.
HIV infection, and especially HIV-1 infection, appears to influence cell
20 surface expression of the leukocyte integrins and cellular adherence reactions
mediated by these heterodimers (Petit, A. J., et ah, J. Clin. Invest. 7P:188
(1987); Hildreth, J.E.K., et al, Science 244:1075 (1989); Valentin, A., et
al, J. Immunology 144:934-937 (1990); Rossen, R.D., et al, Trans. Assoc.
American Physicians 702:117-130 (1989), all of which references are
25 incorporated herein by reference). Following infection with HTV-1,
homotypic aggregation of U937 cells is increased, as is cell surface expression
of CD18, CDllb (Petit, A.J., etal, J. Clin. Invest. 7P:188 (1987)). HIV-1
infected U937 cells adhere to IL-1 stimulated endothelium in greater frequency
than uninfected U937 cells; this behavior can be suppressed by treating the
30 infected cells with anti-CD 18 or anti-CD 11a monoclonal antibodies or by
treating endothelial substrates with anti-ICAM-1 (Rossen, R.D., etaL, Trails.
WO 91/16928
PCT/US91/02946
- 10-
Assoc. American Physicians 102:117-130 (1989)). Monoclonal antibodies to
CD18 or CDlla have also been found to be able to inhibit formation of
syncytia involving phytohemagglutinin (PHA)-stimulated lymphoblastoid cells
and constitutively infected, CD4-negative T cells (Hildreth, J.E.K. , et al ,
5 Science 244:1075 (1989)). Treatment of only the virus infected cells with
anti-CD18, or anti-CDlla monoclonal antibodies was found to have little
effect on syncytium formation, suggesting that these antibodies principally
protect uninfected target cells from infection (Hildreth, J.E.K. , et al, Science
244:1075 (1989); Valentin, A., et al, /. Immunology 144:934-937 (1990)).
10 Valentin et al. (Valentin, A. , et al , J. Immunology 144:934-937 (1990)) have
recently confirmed these observations by demonstrating that monoclonal
antibodies specific for CD 18 inhibit syncytia formed when continuous T cell
lines are co-cultured with HIV-1 infected U937 cells.
Although the mechanism through which monoclonal antibodies specific
15 for CD18 or CDlla protect susceptible cells from fusing with HIV infected
cells remains unknown, and is not necessary to an appreciation of the present
invention, studies with radiolabeled gpl20 suggest that heterodimers
containing CD18 do not provide a binding site for the virus (Valentin, A., et
al, J. Immunology 144:934-937 (1990)). Thus, HIV infection involves cell-
20 cell interactions, and/or viral-cell interactions which mimic such cell-cell
interactions. The cell-cell interactions may result in the transport of cell-free
virus or the transport of virus across endothelial barriers within the cytoplasm
of infected mononuclear cells. Viral-cell interactions which mimic the cell-
cell interactions may facilitate or enable free virus to attach to and/or infect
25 healthy cells.
The present invention thus derives, in part, from the observation that
HIV infection, and particularly HIV-1, infection results in increased
expression of the CDlla/CD18 heterodimer, and its binding ligand, ICAM-1.
This increased expression is significant in that it enhances the ability of HIV-
infected T cells to adhere or aggregate with one another (i.e. to undergo
"homotypic aggregation"). Since such homotypic aggregation is not observed
30
WO 91/16928
PCT/US91/02946
- 11 -
to occur among quiescent normal leukocytes, this discovery indicates that the
expression of the CD11/CD18 receptors and/or ICAM-1 is required for such
aggregation. Such adhesion permits HIV-1 to be transmitted from an infected
cell to a healthy cell of an individual, and also permits or facilitates infection
5 of healthy cells with free virus.
Since ICAM-1 plays a central role in cell-cell interactions murine
monoclonal antibodies that bind to ICAM-1 have been proposed as a method
of preventing HIV infection (WO 90/13281).
F. Migration of HIV Infected Cells
10 The migration and dissemination of leukocytes is important in
protecting an individual from the consequences of infection. These processes,
however, are also responsible for the migration and dissemination of viral-
infected leukocytes. Of particular concern is the migration and dissemination
of leukocytes infected with HIV. The migration of such cells results in the
15 formation of extravascular foci, and may cause tumors and other
abnormalities.
Histologic examination of affected organs reveals focal extravascular
mononuclear cell infiltrates. Attempts to identify virus-infected cells in such
infiltrates in the central nervous system have revealed the presence of HIV-1
20 infected cells. These studies have shown that HIV-1 resides primarily in
monocytes and macrophages, and other cells of this lineage (R.T. Johnson, et
al. FASEB J. 2:2970 (1988); M.H. Stoler et al., J. Amer. Med. Assn.
255:2360 (1986); S. Gartner et al. J. Amer. Med. Assn. 255:2365 (1986); S.
Gartner et al. Science 233:215 (1986)).
25 The mechanisms which stimulate formation of extravascular infiltrates
of HIV-1 -infected monocytoid cells have not previously been well defined.
The mechanisms may involve either the transport of cell-free virus or the
transport of virus across endothelial barriers within the cytoplasm of infected
mononuclear cells.
WO 91/16928
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- 12 -
Since infection with HIV-1 stimulates cell surface expression of
molecules which facilitate adherence of leukocytes to vascular endothelial cells
and the translocation of leukocytes from the blood to extravascular tissue sites
(C.W. Smith et al, J. Clin. Invest. 82:1746 (1988), herein incorporated by
5 reference) it has been proposed to use antibodies which inhibit cellular
migration to prevent the dissemination of HIV infected cells (WO 90/13316).
G. Asthma: Clinical Characteristics
Asthma is a heterogeneous family of diseases. It is characterized by
a hyper-responsiveness of the tracheobronchi to stimuli (McFadden, E.R. et
10 al , In: Harrison 9 s Principles of Internal Medicine, 10th Ed. , Petersdorf , R.G.
et al, Eds., McGraw-Hill, NY (1983), pages 1512-1519); Kay, A.B., Allergy
and Inflammation, Academic Press, NY (1987); which references are
incorporated herein by reference). Clinically, asthma is manifested by the
extensive narrowing of the tracheobronchi, by thick tenacious secretions, by
15 paroxysms of dyspnea, cough, and wheezing. Although the relative
contribution of each of these conditions is unknown, the net result is an
increase in airway resistance, hyperinflation of the lungs and thorax, abnormal
distribution of ventilation and pulmonary blood flow. The disease is
manifested in episodic periods of acute symptoms interspersed between
20 symptom-free periods. The acute episodes result in hypoxia, and can be fatal.
Approximately 3 % of the general world population suffers from the disease.
Two types of asthma have been described: allergic asthma and
idiosyncratic asthma. Allergic asthma is usually associated with a heritable
allergic disease, such as rhinitis, urticaria, eczema, etc. The condition is
25 characterized by positive wheal-and-flare reactions to intradermal injections
of airborne antigens (such as pollen, environmental or occupational pollutants,
etc.), and increased serum levels of IgE. The development of allergic asthma
appears to be causally related to -the presence of IgE antibodies in many
patients. Asthma patients who do not exhibit the above-described
30 characteristics are considered to have idiosyncratic asthma.
WO 91/16928
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Allergic asthma is believed to be dependent upon an IgE response
controlled by T and B lymphocytes and activated by the interaction of airborne
antigen with mast cell-bound pre-formed IgE molecules. The antigenic
encounter must occur at concentrations sufficient to lead to IgE production for
5 a prolonged period of time in order to sensitize an individual. Once
sensitized, an asthma patient may exhibit symptoms in response to extremely
low levels of antigen.
Asthma symptoms may be exacerbated by the presence and level of the
triggering antigen, environmental factors, occupational factors, physical
10 exertion, and emotional stress.
Asthma may be treated with methylxanthines (such as theophylline),
beta-adrenergic agonists (such as catecholamines, resorcinols, saligenins, and
ephedrine), glucocorticoids (such as hydrocortisone), inhibitors of mast cell
degranulation (i.e. chromones such as cromolyn sodium) and anticholinergics
IS (such as atropine).
Asthma is believed to involve an influx of eosinophils ("eosinophilia")
into the tissues of the lung (Frigas, E. et ah, J. Allergy Clin. Immunol.
77:527-537 (1986), which reference is incorporated herein by reference).
Insight into the immunological basis of asthma has been gained from
20 bronchoalveolar lavage studies (Godard, P. et al. , J. Allergy Clin. Immunol.
70:88 (1982)), and studies of respiratory smooth muscle denuded of
epithelium (Flavahan, N.A. et al. 9 J. Appl. Physiol. 58:834 (1985); Barnes,
P. J. et al, Br. J. Pharmacol. 56:685 (1985)). Although these studies have
not led to the elucidation of the mechanism underlying the immunology of
25 asthma, they have led to the development of a generally accepted hypothesis
concerning the immunological etiology of the disease (see, Frigas, E. et al. ,
J. Allergy Clin. Immunol. 77:527-537 (1986)).
The hallmarks of the pathology of asthma are a massive infiltration of
the lung parenchyma by eosinophils and the destruction of mucociliary
30 capacity. The "eosinophil hypothesis" suggests that eosinophils are attracted
to the bronchus in order to neutralize harmful mediators released by the mast
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cells of the lung. According to the hypothesis eosinophils are attracted to the
bronchi where they degranulate to release cytotoxic molecules. Upon
degranulation, eosinophils release enzymes such as histaminase, arylsulfatase
and phospholipase D which enzymatically neutralize the harmful mediators of
5 the mast cell. These molecules also promote the destruction of the
mucociliary apparatus, and thus prevent the clearing of the bronchial
secretions, and contribute to the lung damage characteristic of asthma.
Since asthma involves the migration of cells, it has been proposed to
use antibodies which inhibit this migration to mitigate the effects of allergens
10 in a subject (WO 90/10453).
H. Conclusion
It has been previously proposed; to treat leucocyte-mediated
inflammation by administering inter alia an anti-ICAM-1 antibody to patients
suffering from such inflammation (see EP-0289949 and EP-0314863), to treat
15 viral infection by administering inter alia an anti-ICAM-1 antibody to patients
suffering from such infection (EP 391088), to prevent the infection of a
subject with HIV by administering inter alia an anti-ICAM-1 antibody (WO
90/13281), to prevent the dissemination of HIV infected cells by administering
inter alia an anti-ICAM-1 antibody (WO 90/13316), and to administer inter
20 alia an anti-ICAM-1 antibody to mitigate the effects of allergens (WO
90/10453).
EP 289949 describes the preparation of a murine monoclonal (R6-5-
D6) having specificity for ICAM-1 which is the preferred antibody for the
above referenced therapies. Samples of R6-5-D6 have been deposited with the
25 American Type Culture Collection as deposit ATCC HB9580 on 30th October
1987. R6-5-D6 has been deposited with the ATCC under the provisions of
Rule 28(4) of the EPC.
Currently available anti-ICAM-1 MAbs, which are the basis of the
above described methods of treatment, are murine MAbs and as a result are
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likely to cause a significant HAMA response if administered in repeat doses
to human patients. It would be highly desirable to diminish or abolish this
undesirable HAMA response by suitable humanization or other appropriate
recombinant DNA manipulation of these potentially highly useful antibodies
5 and thus extend and enlarge their use. It would also be desirable to apply the
techniques of recombinant DNA technology to these antibodies to prepare
anti-ICAM-1 humanized chimeric antibodies in general.
We have now prepared anti-ICAM-1 chimeric humanized antibody
molecules derived from murine MAbs.
10 SUMMARY OF THE INVENTION
The present invention provides a method of constructing a chimeric
antibody molecule. Specifically the present invention provides a chimeric
antibody molecule comprising heavy and/or light chain variable regions of an
anti-ICAM-1 antibody.
15 The invention further pertains to the chimeric antibody of the present
invention which are detectably labeled.
The present invention further provides a process for producing an anti-
ICAM-1 humanized chimeric antibody molecule.
The present invention further provides DNA coding for a heavy or
20 light chain variable region of a chimeric anti-ICAM-1 antibody.
The invention additionally includes a recombinant DNA molecule
capable of expressing the chimeric antibodies of the present invention.
The invention further includes a host cell capable of producing the
chimeric antibodies of the present invention when transformed by the
25 recombinant DNA molecules disclosed herein.
The invention additionally includes diagnostic and therapeutic uses for
the chimeric antibodies of the present invention.
The invention further provides a method for treating inflammation
resulting from a response of the specific defense system in a mammalian
- 16-
subject which comprises providing to a subject in need of such treatment an
amount of an anti-inflammatory agent sufficient to suppress the inflammation,
wherein the anti-inflammatory agent is a humanized chimeric antibody capable
of binding to ICAM-1.
The invention further provides a method for treating non-specific
inflammation in humans, and other mammals.
In detail, the invention includes a method for treating inflammation
resulting from a response of the specific and non-specific defense system in
a mammalian subject which comprises providing to a subject in need of such
treatment an anti-inflammatory agent, capable of binding to an ICAM-1, in an
amount sufficient to suppress the inflammation; wherein the anti-inflammatory
agent is a humanized chimeric antibody capable of binding to ICAM-1.
The invention further includes the above-described method for treating
inflammation wherein the inflammation is associated with a condition selected
from the group consisting of: adult respiratory distress syndrome; multiple
organ injury syndrome secondary to septicemia; multiple organ injury
syndrome secondary to trauma; reperfusion injury of myocardial or other
tissues; acute glomerulonephritis; reactive arthritis; dermatosis with acute
inflammatory components; acute purulent meningitis or other central nervous
system inflammatory disorders such as stroke; thermal injury; hemodialysis;
leukapheresis; ulcerative colitis; Crohn's disease; necrotizing enterocolitis;
granulocyte transfusion associated syndrome; and cytokine-induced toxicity.
The invention further provides a method of suppressing the metastasis
of a hematopoietic tumor cell, the cell requiring a functional member of the
LFA-1 family for migration, wherein said method comprises providing to a
patient in need of such treatment an amount of an anti-inflammatory agent
sufficient to suppress the metastasis; wherein the anti-inflammatory agent is
a humanized chimeric antibody capable of binding to ICAM-1.
The invention further provides a method of suppressing the growth of
an IC AM- 1 -expressing tumor cell which comprises providing to a patient in
need of such treatment an amount of a toxin sufficient to suppress the growth,
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the toxin being derivatized to one of the chimeric antibodies of the present
invention.
The invention further provides a method of diagnosing the presence
and location of an inflammation resulting from a response of the specific
5 defense system in a mammalian subject suspected of having the inflammation
which comprises:
(a) administering to the subject a composition containing a
detectably labeled chimeric antibody capable of identifying a cell which
expresses ICAM-1, and
10 (b) detecting the binding ligand.
The invention additionally provides a method of diagnosing the
presence and location of an inflammation resulting from a response of the
specific defense system in a mammalian subject suspected of having the
inflammation which comprises:
15 (a) incubating a sample of tissue of the subject with a composition
containing a detectably labeled chimeric antibody capable of identifying a cell
which expresses ICAM-1, and
(b) detecting the binding ligand.
The invention further provides a method of diagnosing the presence
20 and location of an ICAM-l-expressing tumor cell in a mammalian subject
suspected of having such a cell, which comprises:
(a) administering to the subject a composition containing a
detectably labeled chimeric antibody capable of binding to ICAM-1, and
(b) detecting the binding ligand.
25 The invention further provides a method of diagnosing the presence
and location of an ICAM-l-expressing tumor cell in a mammalian subject
suspected of having such a cell, which comprises:
(a) incubating a sample of tissue of the subject with a composition
containing a detectably labeled chimeric antibody capable of binding ICAM-1,
30 and
(b) detecting the binding ligand.
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The invention additionally includes a pharmaceutical composition
comprising:
(a) an anti-inflammatory agent consisting of a chimeric antibody
capable of binding to ICAM-1, and
5 (b) at least one immunosuppressive agent selected from the group
consisting of: dexamethasone, azathioprine and cyclosporin A.
The present invention also relates to the use of chimeric antibodies
capable of binding ICAM-1 in anti-viral therapy.
In detail, the invention provides a method for treating viral infection,
10 wherein said virus binds to the ICAM-1 receptor, in an individual in need of
such treatment, wherein the method comprises providing to the individual an
amount of a chimeric antibody capable of binding ICAM-1 sufficient to
suppress viral infection.
The invention further provides a method for suppressing the infection
15 of leukocytes with HIV, which comprises administering to a patient exposed
to or effected by HIV, an effective amount of an HIV-1 infection suppression
agent, the agent being a chimeric antibody capable of binding to ICAM-1.
The invention further concerns the embodiment of the above method
wherein the HIV is HTV-1.
20 The invention further provides a method for suppressing the
extravascular migration of a virally infected leukocyte in a patient having such
a leukocyte, which comprises administering to the patient an effective amount
of a chimeric antibody capable of impairing the ability of said leukocyte to
bind to ICAM-1.
25 The invention further comprises the embodiment of the above-described
method wherein the virally infected leukocytes are infected with HIV.
The invention further comprises the embodiment of the above-described
method wherein the agent is a chimeric antibody capable of binding to ICAM-
1.
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The invention further provides a method for treating asthma in a
patient which comprises providing to the patient an effective therapeutic
amount of a chimeric antibody capable of binding to ICAM-1
The invention further concerns the embodiment of the above described
5 methods wherein the chimeric antibody capable of binding to ICAM-1 is
derived from the murine monoclonal antibody R6-5-6D.
Brief Description of the Figures
Figure 1 shows the cDNA sequence for the 5' untranslated region, signal
sequence, variable region and part constant region for the R6-
10 5-D6 murine MAb light chain;
Figure 2 shows similar cDNA and amino acid sequence for the R6-5-D6
murine MAb heavy chain;
Figure 3 shows a plasmid diagram of plasmid expression vector pEE6
hCMV;
15 Figure 4 shows plasmid diagrams indicating the strategy for construction
of light chain expression plasmid pAL5;
Figure 5 shows plasmid diagrams indicating the strategy for construction
of heavy chain expression plasmid pAL6;
Figure 6 shows a graph giving results of a competition assay comprising
20 binding of recombinant and murine R6-5-D6 and a control
MAb UPC10;
Figure 7 shows plasmid diagrams indicating the strategy for the
construction of chimeric light chain expression vector pAL7;
Figure 8 shows plasmid diagrams indicating the strategy for the
25 construction of chimeric heavy chain (IgG2 isotype) expression
vector pAL8;
Figure 9 shows outline restriction maps for the chimeric heavy chain
expression vectors pAL8 and pAL9;
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Figure 10 shows plasmid diagrams indicating the procedures involved in
the construction of the GS amplification chimeric light chain
expression vector pALlO;
Figure 11 shows similar plasmid diagrams for the construction of the GS
5 chimeric heavy chain (IgG2 isotype) expression vector pAL12;
Figure 12 shows similar diagrams for the chimeric heavy chain (IgG4
isotype);
Figure 13 shows SDS-PAGE analysis under non-reducing and reducing
conditions. The notation above each lane describes the type of
10 gene used in the transient expression experiment.
mL mouse light cL chimeric light
7 4 chimeric y 4 heavy mH mouse heavy
7 2 chimeric y 2 heavy B72.3 control cL/cH genes
Figure 14 shows SDS-PAGE analysis of purified chimeric anti-ICAM-1
15 antibody or (A) non-reducing and (b) reducing gels.
On each gel Lane 1 is control chimeric B72.3
antibody (IgG4)
Lane 2 is Pharmacia low molecular
weight markers
20 Lanes 3-9 are chimeric anti-ICAM IgG2
4(ig -* 0.125/ig in doubling
dilutions
Lane 10 is Pharmacia low molecular
weight markers
25 Lanes 11-17 are chimeric anti-ICAM IgG4
4/ig -* 0.125/ag in doubling
dilutions
Figure 15 shows HPLC Gel filtration of chimeric anti-ICAM antibody of
IgG2 and IgG4 isotypes.
30 The profiles are superimposable and elute at a time which
corresponds to 150kd tetrameric antibody.
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Figures 16 shows graphs of binding assays of chimeric
and 17 antibodies against standards, and
Figures 18 shows graphs of competition binding assays
19 and 20 of chimeric antibodies against standards.
Figure 21 Inhibition of MLR with antibodies.
Figure 22 Inhibition of Vascular permeability in a modified
Schwartzmann Assay with antibodies.
Brief Description of the Preferred Kmhodimenfs
A. Humanized Antibodies
10 The first embodiment of the present invention provides a chimeric
antibodies molecule comprising heavy and/or light chain variable regions of
an anti-ICAM-1 antibody.
Typically the anti-ICAM-1 antibody is a rodent MAb.
Specifically, in the first embodiment of the present invention the
IS chimeric antibody is a humanized chimeric antibody molecule. In which case
the chimeric antibody comprises heavy and/or light chain variable regions of
a non-human (e.g. , rodent) anti-ICAM-1 antibody linked to heavy and/or light
chain constant region domains of a human antibody. The DNA which codes
for such humanized chimeric heavy and/or light chains comprises DNA coding
20 for the non-human variable region domains linked to DNA coding for human
constant region domains.
The chimeric antibodies molecule of the present invention may
comprise: a complete antibody molecule, having full length heavy and light
chains; a fragment thereof, such as the Fab or (Fab') 2 fragment; a light chain
25 or heavy chain monomer or dimer, including fragments thereof or any
chimeric antibody molecule with the same specificity as an anti-ICAM-1
antibody.
The chimeric antibodies of the present invention may be a "chemical
derivative" of the antibody. As used herein, a molecule is said to be a
-22-
"chemical derivative" of another molecule when it contains additional
chemical moieties not normally a part of the molecule. Such moieties may
improve the molecule's solubility, absorption, biological half life, etc. The
moieties may alternatively decrease the toxicity of the molecule, eliminate or
attenuate any undesirable side effect of the molecule, etc. Moieties capable
of mediating such effects are disclosed in Remington's Pharmaceutical
Sciences (1980). "Toxin-derivatized" molecules constitute a special class of
"chemical derivatives. " A "toxin-derivatized" molecule is a molecule (such
as ICAM-1 or an antibody) which contains a toxin moiety. The binding of
such a molecule to a cell brings the toxin moiety into close proximity with the
cell and thereby promotes cell death. Any suitable toxin moiety may be
employed; however, it is preferable to employ toxins such as, for example,
the ricin toxin, the diphtheria toxin, radioisotopic toxins, membrane-channel-
forming toxins, etc. Procedures for coupling such moieties to a molecule are
well known in the art. Alternatively the chimeric antibody can be attached to
a macrocycle, for chelating a heavy metal atom.
Alternatively, the procedures of recombinant DNA technology may be
used to produce a "chemical derivative" of the chimeric antibody in which the
Fc fragment or CH3 domain of a complete antibody molecule has been
replaced by or has attached thereto by peptide linkage a functional
non-immunoglobulin protein such as an enzyme or toxin molecule.
In the case of humanized chimeric antibody molecules, the remainder
of the molecule may be derived from any suitable human immunoglobulin.
Human constant region domains may be selected having regard to the
proposed function of the antibody in particular the effector functions which
may be required. For example, the constant region domains may be human
IgA, IgE, IgG or IgM domains. In particular, IgG human constant region
domains may be used including any of the IgGl, IgG2, IgG3 and IgG4
isotypes. Thus IgG2 or preferably IgG4 isotypes may be used when the
humanized chimeric antibody is intended for therapeutic purposes, requiring
an absence of antibody effector functions e.g., to block ICAM-l-LFA-1
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interactions. Chimeric IgG anti-ICAM-1 antibodies may have different
avidities depending on i so type and this may influence the therapeutic choice.
Most preferably IgGl human constant region domains are used. We have
found that a IgGl chimeric antibody appears to have a higher binding avidity
5 for ICAM-1 than IgG2 or IgG4 chimeric antibodies, possibly due to a greater
flexibility of the IgGl hinge promoting bivalent binding to the antigen.
The remainder of the humanized chimeric antibody molecule need not
comprise only protein sequences from the human immunoglobulin. For
instance, a gene may be constructed in which a DNA sequence encoding part
10 of a human immunoglobulin chain is fused to a DNA sequence encoding the
amino acid sequence of a polypeptide effector or reporter molecule.
Preferably, the chimeric antibody molecule of the present invention
will be produced by recombinant DNA technology.
A second embodiment of the present invention provides DNA coding
15 for a heavy or light chain variable region of an anti-ICAM-1 antibody.
A third embodiment of the present invention provides a process for
producing an anti-ICAM-1 humanized chimeric antibody molecule which
process comprises:
(a) producing in an expression vector an operon having a DNA
20 sequence which encodes an antibody heavy or light chain
wherein at least the variable domain is derived from a non-
human (rodent) anti-ICAM-1 antibody and the remaining
immunoglobulin-derived parts of the antibody chain are derived
from a human immunoglobulin;
25 (b) producing in an expression vector an operon having a DNA
sequence which encodes a complementary antibody light or
heavy chain wherein at least the variable domain is derived
from a non-human- (rodent) anti-ICAM-1 antibody and the
remaining immunoglobulin-derived parts of the antibody chain
30 are derived from a human immunoglobulin;
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(c) transfecting a host cell with the or each vector; and
(d) culturing the transfected cell line to produce the humanized
chimeric antibody molecule.
The cell line may be transfected with two vectors, the first vector
5 containing an operon encoding a light chain-derived polypeptide and the
second vector containing an operon encoding a heavy chain-derived
polypeptide. Preferably, the vectors are identical except in so far as the
coding sequences and selectable markers are concerned so as to ensure as far
as possible that each polypeptide chain is equally expressed.
1° Alternatively, a single vector may be used, the vector including the
sequences encoding both light chain- and heavy chain-derived polypeptides.
The DNA in the coding sequences for the light and heavy chains may
comprise cDNA or genomic DNA or both. However, it is preferred that the
DNA sequence encoding the heavy or light chain comprises at least partially
15 genomic DNA. Most preferably, the heavy or light chain encoding sequence
comprises a fusion of cDNA and genomic DNA.
Thus, the present invention also includes cloning and expression
vectors and transfected cell lines used in the process of the invention.
The general methods by which the vectors may be constructed,
20 transfection methods and culture methods are well known per se and form no
part of the invention. Such methods are shown, for instance, in Maniatis
etaL, Molecular Cloning, Cold Spring Harbor, New York (1982); and
Primrose and Old, Principles of Gene Manipulation, Blackwell, Oxford
(1980).
25 The anti-ICAM-1 antibodies of the invention include all anti-ICAM-1
specificities. Typically, however, the antibodies have specificity for antigenic
epitopes of ICAM-1 which when bound by the antibody block, inhibit or
otherwise modify ICAM-l/LFA-1 and or ICAM-l/Mac-1 interactions.
Preferably the antibodies have specificity for the same or similar ICAM-1
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antigenic epitopes as the R6-5-D6 etc. antibodies. Most especially the
antibodies are derived from the R6-5-D6 antibody.
B. Therapeutics and Diagnosis
The present invention also includes therapeutic and diagnostic
5 compositions containing the chimeric antibodies of the invention and uses of
such compositions in therapy and diagnosis.
The therapeutic uses to which the products of the anti-ICAM-1
invention may be put include any of the therapeutic uses to which anti-ICAM-
1 antibodies may be put including for example any or all of the therapeutic
10 uses described in EP-0289949, EP-0314863, and corresponding applications.
1. Anti-Inflammatory Agents
A. Specific Inflammation
Monoclonal antibodies to members of the CD 18 or CD-I 1/18 complex
inhibit many adhesion dependent functions of leukocytes including binding to
15 endothelium (Haskard, D., et al, J. Immunol 757:2901-2906 (1986)),
homotypic adhesions (Rothlein, R., et al, J. Exp. Med. 155:1132-1149
(1986)), antigen and mitogen induced proliferation of lymphocytes (Davignon,
D., et al, Proc. Natl Acad. Scl, USA 78:4535-4539 (1981)), antibody
formation (Fischer, A., et al, J. Immunol 756:3198-3203 (1986)), and
20 effector functions of all leukocytes such as lytic activity of cytotoxic T cells
(Krensky, A.M., et al, J. Immunol 752:2180-2182 (1984)), macrophages
(Strassman, G., et al, J. Immunol 755:4328-4333 (1986)), and all cells
involved in antibody-dependent cellular cytotoxicity reactions (Kohl, S.,etal,
J. Immunol 755:2972-2978 (1984)). In all of the above functions, the
25 antibodies inhibit the ability of the leukocyte to adhere to the appropriate
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cellular substrate which in turn inhibits the final outcome. Although both
polyclonal and monoclonal antibodies may be employed to inhibit these
function, the present invention provides an improvement through the use of
a chimeric anti-ICAM-1 antibody.
5 As discussed previously, the binding of ICAM-1 molecules to the
members of LFA-1 family of molecules is of central importance in cellular
adhesion. Through the process of adhesion, lymphocytes are capable of
continually monitoring an animal for the presence of foreign antigens.
Although these processes are normally desirable, they are also the cause of
10 organ transplant rejection, tissue graft rejection and many autoimmune
diseases. Hence, any means capable of attenuating or inhibiting cellular
adhesion would be highly desirable in recipients of organ transplants, (e.g.,
kidney), tissue grafts or autoimmune patients.
A chimeric antibody capable of binding to ICAM-1 is highly suitable
15 as an anti-inflammatory agent in a mammalian subject. Significantly, such an
agent differs from general anti-inflammatory agents and non-humanized
antibodies in that they are capable of selectively inhibiting adhesion, do not
offer other side effects such as nephrotoxicity which are found with conven-
tional agents, and limit the amount of HAMA associated with the use of
20 murine MAbs. A chimeric antibody capable of binding to ICAM-1 can
therefore be used to prevent organ (e.g. kidney) or tissue rejection, or modify
autoimmune responses without the fear of such side effects, in the mammalian
subject.
Importantly, the use of humanized antibodies capable of recognizing
25 ICAM-1 may permit one to perform organ transplants even between
individuals having HLA mismatch.
In the forth embodiment of the present invention a method for
suppressing specific inflammation is provided wherein said method comprises
providing to recipient subjects in need of such a treatment an amount of one
30 of the chimeric antibodies of the present invention sufficient to suppress
inflammation. An amount is said to be sufficient to H suppress M inflammation
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if the dosage, route of administration, etc. of the agent are sufficient to
attenuate or prevent inflammation.
The chimeric antibody may be administered either alone or in
combination with one or more additional immunosuppressive agents
5 (especially to a recipient of an organ (e.g. kidney) or tissue transplant). The
administration of such a composition may be for either a M prophylactic H or
M therapeutic H purpose. When provided prophylactically, the
immunosuppressive composition is provided in advance of any inflammatory
response or symptom (for example, prior to, at, or shortly after) the time of
10 an organ or tissue transplant but in advance of any symptoms of organ
rejection). The prophylactic administration of the composition serves to
prevent or attenuate any subsequent inflammatory response (such as, for
example, rejection of a transplanted organ or tissue, etc.). When provided
therapeutically, the immunosuppressive composition is provided at (or shortly
15 after) the onset of a symptom of actual inflammation (such as, for example,
organ or tissue rejection). The therapeutic administration of the composition
serves to attenuate any actual inflammation (such as, for example, the
rejection of a transplanted organ or tissue).
The anti-inflammatory agents of the present invention may, thus, be
20 provided either prior to the onset of inflammation (so as to suppress an
anticipated inflammation) or after the initiation of inflammation.
Since ICAM-1 molecules are expressed mostly at sites of inflammation,
such as those sites involved in delayed type hypersensitivity reaction,
antibodies (especially chimeric antibodies derived from anti-ICAM-1
25 monoclonal antibodies) capable of binding to ICAM-1 molecules have
therapeutic potential in the attenuation or elimination of such reactions. This
potential therapeutic use may be exploited in either of two manners. First, a
composition containing a chimeric antibody capable of binding to ICAM-1
may be administered to a patient experiencing delayed type hypersensitivity
30 reaction. For example, such compositions might be provided to a individual
who had been in contact with antigens such as poison ivy, poison oak, etc.
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-28-
In a sixth embodiment, one of the chimeric antibodies of the present
invention is administered to a patient in conjunction with an antigen in order
to prevent a subsequent inflammatory reaction. Thus, the additional
administration of an antigen with an ICAM-1 -binding chimeric antibody may
5 temporarily tolerize an individual to subsequent presentation of that antigen.
Since LAD patients that lack LFA-1 do not mount an inflammatory
response, it is believed that antagonism of LFA-l's natural ligand, ICAM-1,
will also inhibit an inflammatory response. The ability of antibodies against
ICAM-1 to inhibit inflammation provides the basis for their therapeutic use in
10 the treatment of chronic inflammatory diseases and autoimmune diseases such
as lupus erythematosus, autoimmune thyroiditis, experimental allergic
encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes
Reynaud's syndrome, rheumatoid arthritis, etc. Such antibodies may also be
employed as a therapy in the treatment of psoriasis. In general, a chimeric
15 antibody capable of binding ICAM-1 may be employed in the treatment of
those diseases currently treatable through steroid therapy.
B. Non-specific Inflammation
The present invention derives from the discovery that ICAM-1 on
endothelial cells binds to the members of the CD18 family of molecules on
granulocytes responsible for mediating granulocyte-endothelial cell adhesion
and that antagonists of ICAM-1 are capable of inhibiting such adhesion. Such
inhibition provides a means for treating general, non-specific tissue inflammation.
Since cellular adhesion is required in order that leukocytes may
migrate to sites of non-specific inflammation and/or carry out various effector
functions contributing to the inflammation, agents which inhibit cellular
adhesion will attenuate or prevent this inflammation. A "non-specific defense
system reaction" is a response' mediated by leukocytes incapable of
immunological memory. Such cells include granulocytes and macrophages.
As used herein, inflammation is said to result from a response of the non-
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specific defense system, if the inflammation is caused by, mediated by, or
associated with a reaction of the non-specific defense system. Examples of
inflammation which result, at least in part, from a reaction of the non-specific
defense system include inflammation associated with conditions such as: adult
5 respiratory distress syndrome (ARDS) or multiple organ injury syndromes
secondary to septicemia or trauma; reperfusion injury of myocardial or other
tissues; acute glomerulonephritis; reactive arthritis; dermatoses with acute
inflammatory components; acute purulent meningitis or other central nervous
system inflammatory disorders, e.g. stroke; thermal injury; hemodialysis;
10 leukapheresis; ulcerative colitis; Crohn's disease; necrotizing enterocolitis;
granulocyte transfusion associated syndromes; and cytokine-induced toxicity.
In a fifth embodiment of the present invention a method of treating
non-specific inflammation is provided wherein said method comprises
providing to a subject in need of such treatment an effective amount of one of
15 the chimeric antibody of the present invention.
2. Diagnostic and Prpgnpsfo Applications
Since ICAM-1 is expressed mostly at sites of inflammation, a chimeric
antibody capable of binding ICAM-1 may be employed as a means of imaging
or visualizing the sites of infection md inflammation in a patient.
20 In an eighth embodiment of the present invention, a chimeric antibody
is detectably labeled, through the use of radioisotopes, affinity labels (such as
biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc and are
provided to a patient to localize the site of infection or inflammation. Proce-
dures for accomplishing such labeling are well known to the art. Clinical
25 application of antibodies in diagnostic imaging are reviewed by Grossman,
H.B., Urol. Clin. North Amer. -75:465-474 (1986)), Unger, B.C. et al,
Invest. Radiol. 20:693-700 (1985)), and Khaw, B.A. et al., Science 209:295-
297 (1980)).
30-
The detection of foci of such detectably labeled antibodies is indicative
of a site of inflammation or tumor development. In one embodiment, this
examination for inflammation is done by removing samples of tissue,
including blood cells, and incubating such samples in the presence of the
detectably labeled antibodies. In a preferred embodiment, this technique is
done in a non-invasive manner through the use of magnetic imaging, fluoro-
giaphy, etc. Such a diagnostic test may be employed in monitoring organ
transplant recipients for early signs of potential tissue rejection. Such assays
may also be conducted in efforts to determine an individual's predilection to
rheumatoid arthritis or other chronic inflammatory diseases.
3. Adjunct to the Introduction of Antigenic Material Administered
for Therapeutic or Diagnostic Purposes
Immune responses to therapeutic or diagnostic agents such as, for
example, bovine insulin, interferon, tissue-type plasminogen activator or
murine monoclonal antibodies substantially impair the therapeutic or diagnostic
value of such agents, and can, in fact, causes diseases such as serum sickness.
Such a situation can be remedied through the use of the chimeric antibodies
of the present invention. In this embodiment, such antibodies would be
administered in combination with the therapeutic or diagnostic agent.
In a ninth embodiment of the present invention the addition of an
effective amount of a chimeric antibody with specificity to ICAM-1 is
administer to a subject in order to prevent the recipient from recognizing the
agent, and therefore prevent the recipient from initiating an immune response
against it. The absence of such an immune response results in the ability of
the patient to receive additional administrations of the therapeutic or diagnostic
agent.
4. Anti-viral usage of chimeric antibodies
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Another aspect of the present invention relates to the discovery of that
ICAM-1 is the cellular receptor of certain viruses, and is thus required in
order for the virus to adhere to and infect human cells (Greve, J.M. et al ,
Cell 55:839-847 (1989); Staunton, D.E. et al, Cell 55:849-853 (1989), both
5 of which references are incorporated by reference herein in their entirety).
In particular, rhinoviruses, and especially rhinoviruses of the major serotype
have been found to be capable of mediating their infection through their
capacity to bind to the ICAM-1 molecules present on cell surfaces.
The tenth embodiment of the present invention is directed toward the
10 use of chimeric antibodies capable of binding ICAM-1 to treat viral infection.
Because such antibodies are capable of blocking the ICAM-1 of endothelial
cells for viral attachment, their administration to a recipient individual results
in the decrease in receptors available for viral binding, and thus decreases the
percentage of viruses which attach and infect the cells of an individual.
15 ICAM-1 has the ability to interact with and bind to viruses, and in
particular, rhinoviruses of the major serotype within the genus Picornaviridae,
group A coxsackieviruses (Colonno, RJ. et al, J. virol. 57:7-12 (1986)) and
Mengo viruses (Rossmann, M.G. et al, Virol 764:373-382 (1988)). This
interaction is mediated by ICAM-1 amino acid residues which are present in
20 domain 1 of the ICAM-1 molecule. Such interactions are assisted, however,
by contributions from amino acids present in domains 2 and 3 of ICAM-1.
Thus, among the preferred chimeric antibodies of this embodiment are
antibodies capable of binding to domains 1, 2, and 3 of ICAM-1. More
preferred are chimeric antibodies capable of binding to domains 1 and 2 of
25 ICAM-1. Most preferred are chimeric antibodies capable of binding domain
1 of
ICAM-1.
The administration of the anti-viral agents of the present invention may
be for either a "prophylactic" or "therapeutic" purpose. When provided
30 prophylactically, the anti-viral agent is provided in advance of any symptom
of viral infection (for example, prior to, at, or shortly after the time of
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infection, but in advance of any symptoms of such infection). The
prophylactic administration of the agent serves to prevent or attenuate any
subsequent viral infection, or to reduce the possibility that such infection will
be contagious to others.
When provided therapeutically, the anti-viral agent is provided at (or
shortly after) the onset of a symptom of actual viral infection (such as, for
example, nasal congestion, fever, etc. The therapeutic administration of the
agent serves to attenuate any actual viral infection.
The anti-viral agents of the present invention may, thus, be provided
either prior to the onset of viral infection (so as to suppress an anticipated
infection) or after the initiation of such infection.
5. Su ppression of HIV Infection and the prevention of the
Dissemination of HIV Infected Cells.
The eleventh embodiment of present invention provides a method for
suppressing the infection of HIV, which comprises administering to an HIV-
infected individual an effective amount of an HTV infection suppression agent.
Although the invention is particularly concerned with a method for the
suppression of HIV-1 infection, it is to be understood that the method may be
applied to any HIV-1 variant (such as, for example, HIV-2) which may infect
cells in a way which may be suppressed by the agents of the present
invention. Such variants are the equivalents of HIV-1 for the purposes of the
present invention.
One aspect of the present invention derives from the recognition that
expression of LFA-1 and, in some cases, ICAM-1, stimulated by HIV
infection, promotes cell-to-cell adherence reactions that can increase the
contact time of infected with uninfected cells, facilitating transfer of virus
from infected to uninfected cells. Thus, chimeric antibodies capable of
binding ICAM-1 are able to suppress infection by HTV, and, in particular, by
HIV-1.
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One means through which molecules which bind to ICAM-1 may
suppress HIV infection is by impairing the ability of the ICAM-1 expressed
by HIV-infected cells to bind to the CD1 1/CD18 receptors of a healthy T cell.
In order to impair the ability of a cell to bind to the CD 1 la/CD 18 receptor,
5 or to the ICAM-1 ligand molecule, it is possible to employ chimeric
antibodies capable of binding to ICAM-1.
The agents of the present invention are intended to be provided to
recipient subjects in an amount sufficient to achieve a suppression of HIV
infection. An amount is said to be sufficient to M suppress H HIV infection if
10 the dosage, route of administration, etc. of the agent are sufficient to attenuate
or prevent such HTV infection. The agents are to be provided to patients who
are exposed to, or effected by HTV infection.
The chimeric antibodies of the present invention may be for either a
"prophylactic" or "therapeutic" purpose in the treatment of HTV infection.
15 When provided prophylactically, the antibody is provided in advance of any
symptom of viral infection (for example, prior to, at, or shortly after) the time
of such infection, but in advance of any symptoms of such infection). The
prophylactic administration of the antibody serves to prevent or attenuate any
subsequent HIV infection. When provided therapeutically, the antibody is
20 provided at (or shortly after) the detection of virally infected cells. The
therapeutic administration of the antibody serves to attenuate any additional
HTV infection.
The agents of the present invention may, thus, be provided either prior
to the onset of viral infection (so as to suppress the anticipated HTV infection)
25 or after the actual detection of such virally infected cells (to suppress further
infection).
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In particular, the invention provides an improved therapy for AIDS,
and an enhanced means for suppressing HTV infection, and particularly HTV-1
infection, which comprises the co-administration of:
(I) ICAM-1, a soluble ICAM-1 derivative, CD11 (either CDlla,
CDllb, or CDllc), a soluble CD11 derivative, CD18, a
soluble CD 18 derivative, or a CD11/CD18 heterodimer, or a
soluble derivative of a CD11/CD18 heterodimer and/or
(II) a chimeric antibody capable of binding to ICAM-1 with
(HI) cell or particle associated CD4 or a soluble derivative of CD4
and/or
(IV) a molecule (preferably an antibody or antibody fragment)
capable of binding to CD4.
In the twelfth embodiment of the present invention also a method for
suppressing the migration of HIV-infected cells is provided wherein said
method comprises administering an effective amount of an anti-migration
agent to an HIV-infected individual.
The anti-migration agents of the present invention include any chimeric
antibody capable of impairing the ability of an HIV-infected T cell to bind to
ICAM-1. Chimeric antibodies which bind to ICAM-1 will suppress migration
by impairing the ability of the ICAM-1 expressed by HIV-infected T cells to
bind to cells expressing a CD11/CD18 receptor. In order to impair the ability
of a cell to bind to the CDlla/CD18 receptor it is possible to employ a
chimeric antibody capable of binding to ICAM-1.
The agents of the present invention are intended to be provided to
recipient subjects in an amount sufficient to suppress the migration of HIV (or
other virally) infected T cells. An amount is said to be sufficient to
"suppress- migration of T cells if the dosage, route of administration, etc. of
the agent are sufficient to attenuate or prevent such migration.
The administration of a chimeric antibody may be for either a
"prophylactic" or "therapeutic" purpose. When provided prophylactically, the
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chimeric antibody is provided in advance of any symptom of viral infection
(for example, prior to, at, or shortly after) the time of such infection, but in
advance of any symptoms of such infection). The prophylactic administration
of the chimeric antibody serves to prevent or attenuate any subsequent
5 migration of virally infected T cells. When provided therapeutically, the
chimeric antibody is provided at (or shortly after) the detection of virally
infected T cells. The therapeutic administration of the antibody serves to
attenuate any additional migration of such T cells.
The antibodies of the present invention may, thus, be provided either
10 prior to the onset of viral infection (so as to suppress the anticipated migration
of infected T cells) or after the actual detection of such virally infected cells.
6. Treatment pf Asthma
In the thirteenth embodiment of the present invention a chimeric
antibody capable of binding to ICAM-1 is used in the treatment of asthma.
15 The therapeutic effects of the anti-asthma agents of the present
invention may be obtained by providing such agents to a patient by any
suitable means (i.e. intravenously, intramuscularly, subcutaneously, enterally,
or parenterally). It is preferred to administer the agents of the present
invention intranasally as by nasal spray, swab, etc. It is especially preferred
20 to administer such agents by oral inhalation, or via an oral spray or oral
aerosol. When administering agents by injection, the administration may be
by continuous infusion, or by single or multiple boluses.
The anti-asthma agents of the present invention are intended to be
provided to recipient subjects in an amount sufficient to lessen or attenuate the
25 severity, extent or duration of the asthma symptoms.
The chimeric antibodies of the present invention may be administered
either alone or in combination with one or more additional anti-asthma agents
(such as methylxanthines (such as theophylline), beta-adrenergic agonists (such
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as catecholamines, resorcinols, saligenins, and ephedrine), glucocorticoids
(such as hydrocortisone), chromones (such as cromolyn sodium) and
anticholinergics (such as atropine), in order to decrease the amount of such
agents needed to treat the asthma symptoms.
The administration of the chimeric antibodies of the present invention
may be for either a "prophylactic" or "therapeutic" purpose. When provided
prophylactically, the chimeric antibodies are provided in advance of any
asthma symptom. The prophylactic administration of the chimeric antibody
serves to prevent or attenuate any subsequent asthmatic response. When
provided therapeutically, the chimeric antibody is provided at (or shortly after)
the onset of a symptom of asthma. The therapeutic administration of the
antibody serves to attenuate any actual asthmatic episode. The antibodies of
the present invention may, thus, be provided either prior to the onset of an
anticipated asthmatic episode (so as to attenuate the anticipated severity,
duration or extent of the episode) or after the initiation of the episode.
C. Administration of the Compositions of the Present Invention
The therapeutic effects of chimeric antibodies capable of binding
ICAM-1 may be obtained by providing to a patient an effective amount of a
chimeric antibody which is substantially free of natural contaminants. The
20 chimeric antibodies of the present invention disclosed herein are said to be
"substantially free of natural contaminants" if preparations which contain them
are substantially free of materials with which these products are normally and
naturally found.
The present invention extends to chimeric antibodies which may be
25 produced either by an animal, or by tissue culture, or recombinant DNA
means.
In providing a patient with a chimeric antibody, the dosage of
administered agent will vary depending upon such factors as the patient's age,
weight, height, sex, general medical condition, previous medical history, etc.
10
15
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In general, it is desirable to provide the recipient with a dosage of antibody
which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of
patient), although a lower or higher dosage may be administered.
A chimeric antibody capable of binding to ICAM-1 may be
5 administered to patients intravenously, intramuscularly, subcutaneously,
enterally , topically inhaled, intranasally, or parenterally. When administering
an antibody, the administration may be by continuous administration, or by
single or multiple boluses.
A composition is said to be "pharmacologically acceptable" if its
10 administration can be tolerated by a recipient patient. Such an agent is said
to be administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the physiology of
a recipient patient.
15 The chimeric antibodies of the present invention can be formulated
according to known methods to prepare pharmaceutical^ useful compositions,
whereby these antibodies are combined in a mixture with a pharmaceutical^
acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive
of other human proteins, e.g., human serum albumin, are described, for
20 example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed.,
Mack, Easton PA (1980)). In order to form a pharmaceutical^ acceptable
composition suitable for effective administration, such compositions will
contain an effective amount of a chimeric antibody together with a suitable
amount of carrier vehicle.
25 Additional pharmaceutical methods may be employed to control the
duration of action. Controlled release preparations may be achieved through
the use of polymers to complex or absorb the chimeric antibody. The
controlled delivery may be exercised by selecting appropriate macromolecules
(for example polyesters, polyamino acids, polyvinyl, pyrrolidone,
30 ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine,
sulfate) and the concentration of macromolecules as well as the methods of
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incorporation in order to control release. Another possible method to control
the duration of action by controlled release preparations is to incorporate the
chimeric antibody into particles of a polymeric material such as polyesters,
poiyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate
5 copolymers. Alternatively, instead of incorporating the antibody into
polymeric particles, it is possible to entrap these materials in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or gelatine-
microcapsules and poly(methylmethacylate) microcapsules, respectively, or in
10 colloidal drug delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and nanocapsules or in
macroemulsions. Such techniques are disclosed in Remington's Pharma-
ceutical Sciences (1980).
MATERIAL, METHODS
Hybridoma cell line R6-5-D6 producing anti-ICAM-1 antibody was
provided by Boehringer Ingelheim Pharmaceuticals Inc. (Lot No. R6-5-D6 -
E9-B2 0-29-86) and was grown up in antibiotic free Dulbecco's Modified
Eagles Medium (DMEM) supplemented with glutamine and 5% foetal calf
20 serum, and divided to provide both an overgrown supernatant for evaluation
and cells for extraction of RNA. The overgrown supernatant was shown to
contain murine IgG2a/kappa antibody. Cell culture supernatant was examined
and confirmed to contain the antibody R6-5-D6.
2. Molecular Biology Procedures
Basic molecular biology procedures were as Maniatis et al (1982)
(Maniatis et al, Molecular Cloning, Cold Spring Harbor, New York (1982))
with, in some cases, minor modifications. DNA sequencing was performed
as described in Sanger et al (1977) (Sanger et al, Proc. Natl Acad. Scl
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USA 74:5463-5467 (1977)) and the Amersham International Pic sequencing
handbook. COS cell expression and metabolic labelling studies were as
described in Whittle et al (1987) (Whittle et al, Prot. Eng. 1, 5:499-505
(1987)). Chinese Hamster Ovary (CHO) transfections and cell culture were
5 performed as described in Gorman (1988) (Gorman, C. , DNA Cloning 2:143-
190 ed. (1988)) and Bebbington and Hentschel (1988) (Bebbington et al , DNA
Cloning 5:163-188 ed. (1988)).
3. Research Assays
3.1. Assay for Secreted Antibody Light Chain
10 Supernatants from CHO cell lines were assayed for secreted light
chain, after transfection with light chain expression vectors, as the first step
in the development of stable cell lines producing whole chimeric antibody.
The procedure was as follows:
96 well microtitre plates were coated with F(ab') 2 goat anti-human kappa light
15 chain. The plates were washed with water and samples added and incubated
for one hour at room temperature. The plates were washed and F(ab')2 goat
anti-human F(ab')2 Horse radish peroxidase (HRPO) conjugate was then added
and incubated for a further hour. Enzyme substrate was then added to reveal
the reaction.
20 3.2. Assembly Assays
Assembly assays were performed on supernatants from transfected
COS cells and from transfected CHO cells to determine the amount of intact
IgG present.
3.2.1 COS and CHO Cells transfected with mouse genes
25 The assembly assay for intact mouse IgG in cell supernatants was an
ELISA with the following format:-
96 well microtitre plates were coated with F(ab') 2 goat anti-mouse IgG Fc.
The pates were washed in water and samples added and incubated for 1 hour
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at room temperature. The plates were washed and F(ab'>2 goat anti-mouse
IgG F(ab') 2 (HRPO conjugated ) was then added. Enzyme substrate was then
added to reveal the reaction. UPC10, a mouse IgG2a myeloma, was used as
a standard.
5 3.2.2 COS and CHO ggUs trpnsfected with Chimftrir f fc nre
The assembly assay for intact humanized anti-ICAM-1 in COS cell
supernatants was an ELISA with the following format:
96 well microtitre plates were coated with F(ab') 2 goat anti-human IgG Fc.
The plates were washed and samples added and incubated for 1 hour at room
10 temperature. The plates were washed and monoclonal mouse anti-human
kappa chain was added and incubated for 1 hour at room temperature. The
plates were washed and F(ab') 2 goat anti-mouse IgG Fc (HRPO conjugated)
was added. Enzyme substrate was then added to reveal the reaction.
Chimeric B72.3 (Bodmer et al, Published International Patent Application
15 WO 89/01783) (IgG4) and pooled, purified human IgG2 and IgG4 (Chemicon)
were used initially as standards. Later, purified chimeric IgG4 anti-ICAM-1
was used is a standard for work with chimeric IgGl anti-ICAM-1. The use
of a monoclonal anti-kappa chain in this assay allows the amount of chimeric
antibody to be read from the standards.
20 3.3. Assay for Antigen Binding Activity
3.3.1 Direct Binding
Material from COS and CHO cell supernatants and purified chimeric
antibodies were assayed for anti-ICAM-1 antigen binding activity onto ICAM-
1 positive cells in a direct assay. The procedure was as follows:
25 JY cells (a human B lymphoblastoid cell line which constitutively expresses
ICAM-1 on the cell surface) were maintained in culture. Monolayers of JY
cells were fixed onto 96 well ELISA plates using poly-L-lysine and
paraformaldehyde, and the plates were blocked with a solution of bovine
serum albumin in PBS. Samples were added to the monolayers and incubated
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for 1 hour at room temperature. The plates were washed gently using PBS.
F(ab') 2 goat anti-human IgG Fc (HRPO conjugated) or F(ab')2 goat anti-mouse
IgG Fc (HRPO conjugated) was then added as appropriate for humanized or
mouse samples. Enzyme substrate was then added to reveal the reaction. The
5 negative control for the cell-based assay was chimeric B72.3 (IgG4) or pooled,
purified human IgG2 and IgG4 (Chemicon). The positive control was murine
R6-5-D6 MAb.
3.3.2 Competit ion Hinging
Monolayers of JY cells were prepared as in 3.3.1. Antibody samples
10 were added and incubated overnight at 4°C. Biotinylated anti-ICAM-1 was
added to all the wells. The mixture was left at room temperature for 2 hours.
The plates were washed and either streptavidin-HRPO or streptavidin-
betagalactosidase was added. After further incubation enzyme substrate was
added to reveal the reaction.
15 3.3.3 Mixed Lymphocyte Reagjjgn Assays
Peripheral blood was obtained from normal, healthy donors by
venipuncture. The blood (7.5 ml) was layered over 7.5 ml of a
Ficoll/Hypaque density gradient (Pharmacia, density = 1.078) room
temperature and centrifuged at 1000 x g for 20 minutes. The cells were
20 washed, counted on a hemacytometer, and suspended in RPMI-1640 culture
medium (Gibco) containing 50 /ig/ml gentamycin, 1 nM L-glutamine (Gibco)
and 5% heat inactivated (55 C,30 min) human AB sera (Flow Laboratories)
(hereafter referred to as RPMI-culture medium).
Peripheral blood mononuclear cells (responder cells) were cultured in
25 medium at 6.25 X 10" 5 cells/ml in Linbro round-bottomed microliter plates
(#76-013-05). Stimulator cells from a separate donor were irradiated at
1000R and cultured with the responder cells at the same concentration.
Responder cells were added to wells, followed by the monoclonal antibodies
and the stimulator cells were added last. The total volume per culture was 0.2
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ml. Controls included responder cells alone. The culture plates were
incubated at 37°C in a 5% C0 2 -humidified air atmosphere for 5 days. The
wells were pulsed with 0.5 uCi tritiated thymidine ^HT) (New England
Nuclear) for the last 18 hours of culture.
5 The cells were harvested onto glass fiber filters using an automated
multiple sample harvester (Skatron, Norway), rinsing with distilled water.
The filters were oven dried and counted in Beckman Ready Safe liquid
scintillation cocktail on a LKB Betaplate liquid scintillation counter.
3.4 In Vivo Assays
10 3.4.1 Modified gfowfl^^ raann Reaction
The local Schwartzmann reaction in rabbit skin can be produced by an
i.d. injection of endotoxin followed by an Lv. challenge injection of zymosan
18-24 hrs. later. The hemorrhagic necrosis that develops in the previously
injected skin sites is characterized by microthrombi, intravascular neutrophil
15 aggregation, platelet and fibrin deposition, vascular permeability increases and
massive extravasation of erythrocytes (RBC). We have modified this protocol
for use in the Cynomologous monkey. Separate, distinct skin sites were
injected i.d. with endotoxin (3 ftg/site) or normal saline and 18 hours later
these same sites were injected with zymosan (300 pig/site). The resulting
20 inflammatory response was quantitated at 6 hours post-zymosan by measuring
the increase in vascular permeability in the endotoxin-injected sites compared
to the saline-injected sites using 125 I-BSA. The inhibitory effect of R6.5 (anti-
ICAM-1), the IgGl, IgG2, and IgG4, chimerics of R6.5 and R15.7 (LFA-1
beta) were compared to that of normal mouse IgG. All IgG preparations were
25 administered i.v. at 3 mg/kg prior to the zymosan injections.
Results
4. CDNA Library Construction
4.1
fflRNA P reparation and cDNA Synthesis
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Cells were grown as described in Section 1 and 1.4 x 10 9 cells
harvested and mRNA extracted using the guanidinium/LiCl extraction
procedure. cDNA was prepared by priming from Oligo-dT to generate full
length cDNA. The cDNA was methylated and EcoRI linkers added for
5 cloning.
4.2 Library CflBStDlCtiflB
The cDNA library was ligated to pSP64 vector DNA which had been
EcoRI cut and the 5' phosphate groups removed by calf intestinal phosphatase
(EcoRI/CIP). The ligation was used to transform high transformation
10 efficiency Escherichia coli HB101 (E. coli HB101) from Bethesda Research
Labs (BRL) in the case of the light chain and E. coli LM1035 prepared by
electroporation (Dower et al, Nucl. Acids Res. 75:6127 (1988)) in the case
of the heavy chain. cDNA libraries were prepared. 11600 colonies were
screened for the light chain and 25000 colonies were screened for the heavy
15 chain.
5. Scrolling
E. coli colonies positive for either heavy or light chain probes were
identified either by oligonucleotide screening using the oligonucleotide: 5'
TCCAGATGTTAACTGCTCAC for the light chain, which is complementary
20 to a sequence in the mouse kappa constant region, or by using a 980 bp
BamHI-EcoRI restriction fragment of a previously isolated mouse IgG2a
constant region clone. 6 light chain and 10 heavy chain clones were identified
and taken for second round screening. Positive clones from the second round
of screening were grown up and DNA prepared. The sizes of the gene inserts
25 were estimated by gel electrophoresis and DNA inserts of a size capable of
containing a full length cDNA were sequenced.
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DNA sequence for the 5' untranslated regions, signal sequences,
variable regions and 3' untranslated regions of full length cDNAs were
obtained and are given in Figure 1 for the light chain and Figure 2 for the
5 heavy chain.
7. Construction of cDNA Expression Vectors
CellTech expression vectors are based on the plasmid pEE6-hCMV as
shown in Figure 3 (Bebbington, C.R., Published International Patent
Application WO 89/01036). A polylinker for the insertion of genes to be
10 expressed has been introduced after the major immediate early
promoter/enhance of the human Cytomegalovirus (hCMV). Marker genes for
selection of the plasmid in transfected eukaryotic cells can be inserted as
BamHI cassettes in the unique BamHI site of pEE6-hCMV. It is usual
practice to insert the neo and gpt markers prior to insertion of the gene of
15 interest, whereas the GS marker is inserted last because of the presence of
internal EcoRI sites in the cassette. The selectable markers are expressed
from the SV40 late promoter which also provides an origin of replication so
that the vectors can be used for expression in the COS cell transient
expression system. The mouse sequences were excised as EcoRI fragments
20 and cloned into either EE6-hCMV-neo for the light chain (Figure 4) and into
EE6-hCMV-gpt for the heavy chain (Figure 5).
8. Expression of cDNAs in COS cells
Plasmids pAL5 (Figure 4) and pAL6 (Figure 5) were co-transfected
into COS cells and supernatant from the transient expression experiment was
25 shown to contain assembled antibody which bound to JY cells (Figure 6).
Metabolic labelling experiments using 35 S methionine showed expression and
assembly of heavy and light chains.
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9. Construction Chimeric ggngs
Construction of chimeric genes followed a previously described
strategy (Whittle etal. 1987, (Whittle et al , Prot. Eng. 1, 5:499-505 (1987)).
A restriction site near the 3' end of the variable domain sequence is identified
5 and used to attach an oligonucleotide adapter which codes for the remainder
of the mouse variable region and includes a suitable restriction site for
attachment to the constant region of choice.
9.1 I4ght Gfoin ggng Cpnstrwtipn
The mouse light chain cDNA sequence showed an SfaNI site near the
10 3* end of the variable region. The majority of the sequence of the variable
region was isolated as a 397 bp. EcoRI-SfaNI fragment. An oligonucleotide
adapter was designed to replace the remainder of the 3' region of the variable
region from the SfaNI site and to include the 5' residues of the human
constant region up to and including a unique Narl site which had been
15 previously engineered into the constant region. The linker was ligated to the
human C K gene in Narl cut pRB32 and the SfaNI-EcoRI adapted C K fragment
was purified from the ligation mixture. The constant region was ligated with
the EcoRI-SfaNI cut variable region DNA into an EcoRI/CIP pEE6-hCMV-
neo treated vector in a three way reaction. Clones were isolated after
20 transformation into E. coli and the linker and junction sequences were
confirmed by DNA sequencing.
Figure 7 shows the strategy for construction of the chimeric light chain.
9.2 Heavy Chain Gene Construction
9.2.1. Choice of Heavy Chain Gene Isotvpe
25 Chimeric heavy chain genes coding for both human IgG2 and IgG4
isotypes were constructed.
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9.2.2. Gene Construction
The heavy chain cDNA sequence showed a BanI site near the 3' end
of the variable region. The majority of the sequence of the variable region
was isolated as a 424bp EcoRI/CIP/BanI fragment. An oligonucleotide
5 adapter was designated to replace the remainder of the 3' region of the
variable region from the BanI site up to and including a unique HindlE site
which had been previously engineered into the first two amino acids of the
constant region. The linker was ligated to the C H gene fragments in HindTTT
cut pRB41 and pRB21 and the Banl-BamHI adapted constant region fragments
10 were purified from the ligation mixture. The EcoRI-BanI variable region
fragment was ligated to each of the constant regions and into the expression
vector (EcoRI/Bcil/CIP treated pEE6-hCMV-gpt) via a three way ligation.
Clones were isolated after transformation into E. coli HB101 and the linker
and junction sequences were confirmed by DNA sequencing. Figure 8 shows
15 the strategy for the construction of the chimeric IgG2 and IgG4 heavy chains
and Figure 9 shows an outline plasmid map of pAL8 (IgG2) and pAL9
(IgG4).
9.2.3 feGl Heavy Chain Gene Construction
Plasmid pElOOl is an expression vector based on pEE6.hCMV gpt.
20 It contains the human IgGl constant region gene. The Apal site which occurs
at the 5th and 6th codon of the Chi domain is unique in this vector, as is a
Hindm site 3' to the hCMV promoter. The VH region of the anti-ICAM-1
heavy chain gene along with the sequence encoding the first five residues of
human CHI was isolated from pAL9 and inserted into pElOOl, previously cut
25 with Hindm and Apal, to give pJA200. The CHI residues carried over from
pAL9 are identical to those for IgGl therefore no novel sequence is generated
at the V-C junction.
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10. Construction of Chimeric Expression Vectors
10.1 GS Separate Vectors
GS versions of pAL7, pAL8 and pAL9 (Figures 7, 8 and 9) were
constructed by replacing the neo and gpt BamHl cassettes with a 5.9Kbp
5 cassette containing the GS gene capable of being expressed from the S V40 late
promoter (See Figures 7, 10-12 for plasmid drawings).
11. Expression Q f Chimeric Gs&£&
11.1 Expression in COS Cells
The chimeric antibody plasmid pAL7 (cL), with either pAL8
10 (cfflgG2), pAL9 (cHIgG4), or pJA200 (CfflgGl) was co-transfected into COS
cells and supernatant from the transient expression experiment was shown to
contain assembled antibody which bound to the JY human B-cell line.
Metabolic antibody which bound to the JY human B-cell line. Metabolic
labelling experiments using 35 S methionine showed expression and assembly
15 of heavy and light chains (Figure 13). All chimeric antibodies bound well to
the JY cells. However, the competition assay showed that antibody derived
from the cL/cHIgG2 or the cL/cHIgG4 combination did not compete as well
as the biotinylated mouse antibody for ICAM-1. The cL/cHIgGl antibody
competed better than the cL/cHIgG2 or cL/cHBIgG4 antibodies.
20 11.2 Expression in Chinese HamStBE Ovarv (CHO) Cells
Stable cell lines were prepared as follows: Chimeric light chain
expression vectors pAL7 and pALlO containing either the neo or GS markers
were transfected into CHO-K1 cells by the CaP0 4 precipitation procedure.
After growth on selective medium, positive cell lines were identified and their
25 specific production rates measured using and ELISA format assay for
detection of secreted light chain. The two cell lines secreting the highest
levels of light chain (see table below) were selected and retransfected with
either pAL8 or pAL9 to introduce the IgG2 and IgG4 chimeric heavy chain
genes along with the gpt marker.
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CELL LINE
SELECTABLE
MARKER
SPECIFIC PRODUCTION
RATE (pg. /cell/day)
24
neo
2.1
46
neo
0.9
25
GS
2.0
| 27
GS
2.3
Around 24 lines shown to be secreting chimeric antibody by assembly assay
ELISA were taken from each transfection. Specific production rates were
measured and the 8 lines with the highest specific production rates are shown
10 below.
The chimeric IgGl heavy chain expression vector pJA200 containing
the gpt marker was transfected into the chimeric light chain expressing cell
line neo24 by the CaP04 precipitation method. Specific production rates were
w
measured, the 4 best lines are shown below.
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1 CELL LINE
SPECIFIC PRODUCTION
RATE (pg. /cell/day)
1 24.1.48
3.75
1 24.2.7
1.2
I 24.1.41
1.3
| 24.2.11
1.75 |
12. Purification of Ch imeric A ntibody
Antibody was purified from 2xlL harvests of supernatant from roller
cultures for cell lines GS 25 G2-12, neo 24 G4-24 and 24. 1.48, and also from
10 2x0.5L harvests from neo 24 G2-9 by affinity chromatography using Protein
A Sepharose. Cell culture supernatants were adjusted to pH8.8 with 0.2M
sodium glycinate and applied to a protein A Sepharose column which had been
pre-equilibrated with glycine/glycinate buffer at pH8. 8. After the samples had
been loaded the column was washed with equilibration buffer. The antibody
IS was then eluted by applying a solution with a decreasing pH gradient
consisting of 0.2M disodium hydrogen phosphate and 0.1M citric acid.
Antibody containing fractions were pooled and the pH adjusted to 6.5 the
samples were then dialyzed against phosphate buffered saline (PBS). Purity
and correct assembly of the antibody was tested by reducing and non-reducing
20 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 14) and by high
performance liquid chromatography (HPLC) gel filtration (Figure 15).
Identity was confirmed by N-terminal amino acid sequencing and amino acid
composition analysis.
13. Analysis of Purified Antibody
25 13.1 Results of Direct Binding and Competitive Binding Assays
All chimeric antibodies bound well to the JY cells (Figures 16 and 17).
However, in competition assays only the cL/cHIgGl antibody competed nearly
as well as the mouse antibody against the biotinylated mouse antibody for
binding to ICAM (Fig. 18). The cL/cfflgG4 antibody showed about 30% of
WO 91/16928
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the inhibitory activity of the mouse antibody (Fig. 19) and the cL/cHIgG2
antibody showed about 10% of the inhibitory activity of the mouse antibody
(Fig. 20).
These data show chimeric anti-ICAM-1 antibodies derived from R6-5-
5 D6 have different antigen binding activities depending on isotype. The IgGl
antibody is nearly as avid as the mouse parent antibody. The IgG4 antibody
as 30% of the competitive binding activity of the mouse antibody, the IgG2
has 10% relative activity. This result is unexpected as all these antibodies
have identical binding sites. We attribute these differences to avidity
10 alterations imposed on the antibodies by the differing hinge flexibility of the
isotypes. Affinity measurements of the chimeric IgG4 Fab have shown that
this has the same affinity as the mouse Fab thus confirming that it is the
avidity that has altered in construction of the chimeric IgG4.
13.2 Results of Mixed Lymphocyte Reaction Assays
15 The MRL which is an in vitro model of transplantation was inhibited
by the chimeric IgG4 and IgGl to comparable extent as the mouse r6-5-6D
antibody. This shows that the chimeric MAb will inhibit specific
immunological events and can thus be used in in vivo autoimmune and
transplantation settings (see Fig. 21).
20 13,3 Results of Modified Schwartzmann Reaction Assays
The primate Schwartzmann reaction was set up to test granulocyte
function in vivo in the presence of an anti-ICAM-1 MAb. The chimeric IgGl
was slightly more active than the mouse MAb which in turn was slightly more
active in inhibiting activated neutrophil mediated vascular leakage than the
25 chimeric IgG4 in the primate model. This demonstrates that the chimeric anti-
ICAM-l's will be effective in mitigating neutrophil mediated damage
associated with reperfusion injury aiid other acute inflammatory disorders (see
Fig. 22).
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CLAIMS;
1. A chimeric antibody molecule comprising heavy and/or light
chain variable regions of an anti-ICAM-1 antibody.
2. The chimeric antibody molecule of claim 1 which is a
5 humanized chimeric antibody.
3. The chimeric antibody molecule of claims 1 or 2 having
attached to it an effector or reporter molecule.
4. The chimeric antibody molecule of claims 1-3 comprising at
least one chimeric heavy chain and at least one chimeric light chain.
10 5. The chimeric antibody of claims 2-4 comprising IgG human
constant region domains.
6. The chimeric antibody of claim 5 comprising IgG2 or IgG4
human constant region domains.
7. The chimeric antibody of claim 5 comprising IgGl human
15 constant region domains.
8. The chimeric antibody of claims 1-7 wherein said chimeric
antibody has binding specificity for the same or similar epitope(s) as the R6-5-
D6 antibody.
9. The chimeric antibody of claims 1-8 wherein said chimeric
20 antibody is derived from the R6-5-D6 antibody.
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10. A DNA sequence encoding the heavy or light chain variable
region of an anti-ICAM-1 antibody.
11. A DNA molecule encoding a chimeric heavy or light chain
comprising the variable region of an anti-ICAM-1 antibody.
5 12. The DNA of claim 11 which codes for a humanized chimeric
heavy or light chain.
13. The DNA of claim 12 coding for a humanized chimeric heavy
chain comprising human IgG constant region domains.
14. The DNA of claim 13 comprising a human IgG2, preferably
10 IgG4 or especially IgGl constant region domains.
15. A vector comprising DNA according to any of claims 10-14.
16. An expression vector comprising in operative combination DNA
coding for a chimeric anti-ICAM-1 light chain and a chimeric anti-ICAM-1
heavy chain.
15 17. A host cell transformed with a vector according to claims 15
or 16.
18. A process for the production of an anti-ICAM-1 humanized
chimeric antibody comprising:
(1) producing an expression vector comprising an operon
20 having a DNA sequence which encodes an antibody heavy or light chain
wherein at least one of the CDRs of the variable domain are derived from a
non-human (rodent) anti-ICAM-1 antibody and the remaining immunoglobulin-
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derived parts of the antibody chain are derived from a human
immunoglobulin;
(2) producing an expression vector comprising an operon
having a DNA sequence which encodes a complementary antibody light or
5 heavy chain wherein at least one of the CDRs of the variable domain are
derived from a rodent (non-human) anti-ICAM-1 antibody and the remaining
immunoglobulin-derived parts of the antibody chain are derived from a human
immunoglobulin;
(3) transfecting a host cell with each vector; and
10 (4) culturing the transfected cell line to produce the chimeric
antibody.
19. A method of treatment comprising administering an effective
amount of an antibody product according to any one of claims 1-9 to a human
or animal subject.
IS 20. A method for treating inflammation resulting from a response
of the specific defense system in a mammalian subject which comprises
providing to a subject in need of such treatment an amount of an anti-
inflammatory agent sufficient to suppress said inflammation, wherein said anti-
inflammatory agent is a chimeric antibody capable of binding ICAM-1.
20 21 . The method of claim 20, wherein said chimeric antibody is one
or more of the antibodies of claims 1-9.
22. The method of claim 20, wherein said inflammation is a delayed
type hypersensitivity reaction.
23. The method of claim 20, wherein said inflammation is a
25 symptom of psoriasis.
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24. The method of claim 20, wherein said inflammation is a
symptom of an autoimmune disease.
25. The method of claim 24, wherein said autoimmune disease is
selected from the group consisting of Reynaud's syndrome, autoimmune
5 thyroiditis, EAE, multiple sclerosis, rheumatoid arthritis and lupus
erythematosus.
26. The method of claim 20, wherein said inflammation is in
response to organ transplant rejection.
27. The method of claim 26, wherein said organ transplant is a
10 kidney transplant.
28. The method of claim 20, wherein said inflammation is in
response to tissue graft rejection.
29. The method of claims 20 which additionally comprises the
administration of an agent selected from the group consisting of: an antibody
15 capable of binding to LFA-1; a functional derivative of said antibody, said
functional derivative being capable of binding to LFA-1; and a non-
immunoglobulin antagonist of LFA-1.
30. A method for treating inflammation resulting from a response
of the non-specific defense system in a mammalian subject which comprises
20 providing to a subject in need of such treatment an amount of an anti-
inflammatory agent sufficient to suppress said inflammation, wherein said anti-
inflammatory agent is a chimeric antibody capable of binding ICAM-1.
*
3 1 . The method of claim 30 wherein said inflammation is associated
with a condition selected from the group consisting of: adult respiratory
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distress syndrome; multiple organ injury syndrome secondary to septicemia;
multiple organ injury syndrome secondary to trauma; reperfusion injury of
tissue; acute glomerulonephritis; reactive arthritis; dermatosis with acute
inflammatory components; a central nervous system inflammatory disorder
5 e.g. stroke; thermal injury; hemodialysis; leukapheresis; ulcerative colitis;
Crohn's disease; necrotizing enterocolitis; granulocyte transfusion associated
syndrome; and cytokine-induced toxicity.
32. The method of claims 30 or 31 wherein said chimeric antibody
is one or more of the antibodies of claims 1-9.
10 33. A method of suppressing the metastasis of a hematopoietic
tumor cell, said cell requiring a functional member of the LFA-1 family for
migration, which method comprises providing to a patient in need of such
treatment an amount of an anti-inflammatory agent sufficient to suppress said
metastasis, wherein said anti-inflammatory agent is a chimeric antibody
15 capable of binding IC AM- 1 .
34. The method of claim 33, wherein said chimeric antibody
capable of binding to ICAM-1 is selected from the group of antibodies of
claims 1-9.
35. A method of suppressing the growth of an IC AM- 1 -expressing
20 tumor cell which comprises providing to a patient in need of such treatment
an amount of a toxin sufficient to suppress said growth, said toxin consists of
a toxin-derivatized chimeric antibody capable of binding to ICAM-1.
36. A method for treating viral infection in an individual in need
of such treatment, wherein said method comprises providing to said individual
56
an amount of a chimeric antibody capable of binding ICAM-1 sufficient to
suppress viral infection.
37. The method of claim 36, wherein said virus is a rhinovirus of
the major serotype within the genus Picornaviridae, a group A coxsackievirus,
or a Mengo virus.
38. The method of claim 37 wherein said virus is a rhinovirus of
the major serotype.
39. The method of any of claims 36-38 wherein said chimeric
antibody is at least one of the antibodies of claims 1-9.
40. A method for suppressing the infection of leukocytes with HIV,
which comprises administering to a patient exposed to or infected by HIV, an
effective amount of an HIV-1 infection suppression agent, said agent being a
chimeric antibody capable of binding to ICAM-1.
41. The method of claim 40 wherein said HIV is HIV-1.
42. The method of any one of claims 40 or 41, wherein said
chimeric antibody is at least one of the antibodies of claims 1-9.
43. A method for suppressing the extravascular migration of a
virally infected leukocyte in a patient having such a leukocyte, which
comprises administering to said patient an effective amount of an anti-
migration agent, said agent being a chimeric antibody capable of impairing the
ability of said leukocyte to bind to ICAM-1.
44. The method of claim 43, wherein said virally infected cells are
infected with HIV.
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45. The method of claims 43 or 44, wherein said chimeric antibody
is at least one of the antibodies of claims 1-9.
46. A method for treating asthma in an individual in need of such
treatment, wherein said method comprises providing to said individual an
5 amount of a chimeric antibody capable of binding ICAM-1 sufficient to
suppress asthma.
47. The method of claim 46 wherein said chimeric antibody is at
least one of the antibodies of claims 1-9.
48. The method of any one of claims 19-47 wherein said humanized
10 chimeric antibody is administered by enteral means, parenteral means, topical
means, inhalation means or intranasal means.
49. The method of claim 48 wherein said humanized chimeric
antibody is administered prophylactically.
50. The method of claim 48 wherein said humanized chimeric
15 antibody is administered therapeutically.
51. The method of claims 48, 49 or 50 wherein said parenteral
means is intramuscular, intravenous or subcutaneous.
52. A pharmaceutical composition comprising the anti-inflammatory
agent of any one of claims 1-9 in combination with a pharmaceutically
20 acceptable carrier.
53. The pharmaceutical .composition of claim 52 in combination
with at least one other immunosuppressive agent.
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54. A method of diagnosing an IC AM- 1 -expressing tumor cell in
a mammalian subject which comprises:
(a) administering to said subject a composition containing
a detectably labeled chimeric antibody capable of binding to ICAM-1, and
5 (b) detecting said chimeric antibody bound to said IC AM- 1 .
55. A method of diagnosing inflammation in a mammalian subject
which comprises:
(a) incubating a sample of tissue of said subject with a
composition containing a detectably labeled chimeric antibody capable of
10 binding to a cell which expresses ICAM-1, and
(b) detecting said chimeric antibody bound to said cell.
WO 91/16928
PCT/US91/02946
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I. CLASSIFICATION OF SU BJECT MATTER M s.wrr.il d.issihc.-.hon symbols .u.fUv. mcnr.Ht.' .»») *
According to International Patent Classii.cat.on (IPC) of 10 both f4at.on.il Ctjss.i.cil.on and IPC
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Category * | Citation
US, A, 4,81.6,567 (CABILY ET AL.) 28 March 1989, See
Entire Document .
JOURNAL OF CLINICAL INVESTIGATION, Vol. 82, issued
November 1988, C.W. Smith et al. , "Recognition of an
Endothelial Determinant for CD18-dependent Human ^
Neutrophil Adherence and Transendothelial Migration ,
pages 1746-1756. See Entire Document.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA,
Vol. 81, issued November 1984, Morrison et al. ,
"Chimeric Human Antibody Molecules: Mouse Antigen-
Binding Domains with Human Constant Region Domains ,
pages 6851-6855. See Entire Document.
NATURE, Vol. 321, issued 29 May 1986, Jones et al. ,
"Replacing the Complementarity-determining regions in
a Human Antibody With Those From a Mouse", pages 522-
525. See Entire Document.
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Oate Ol thf Avlu.il LoiiM.l. l.un Of !»••• tniwn il. .in H
18 JULY 1991
() ill* III *•• IM.II-I wl H • » I Mflll ll.»»H H S." II • '« Hi t ♦ » • * t
16 AUG 1991
I'iIim n.ilioiKii Ajifiin
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■ II. OOCUMENTS CONSIDERED TO CE RELEVANT (CONTINUED TROM THE SECOND SHE ET)
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! R. l.w.int to Cl.nni No
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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, USA,
Vol. 86, issued December 1989, Queen et al. , "A
Humanized Antibody That Binds to the Interleukin-2
Receptor", pages 10029-10033, See Entire Document.
JOURNAL OF IMMUNOLOGY, Vol. 143, No. 4, issued
15 August 1989, Barton et al. , "The Effect of Anti-
Intercellular Adhesion Molecule-1 on Phrobol-Ester-
Induced Rabbit Lung Inflammation, pages 1278-1282,
See Abstract and Discussion.
EUROPEAN JOURNAL OF IMMUNOLOGY, Vol. 20, issued
December 1990, Geissler et al. , "A Monoclonal Antibod-
Directed Against the Human InterCellular Adhesion
Molecule (ICAM-1) Modulates the Release of Tumor
Necrosis Factor-x, Interferon-* and Interleukin-1 ,
pages 2591-2596. See Entire Document.
W0, A, 89/01783, (CELLTECH) 09 March 1989, See
Abstract.
EUROPEAN JOURNAL OF IMMUNOLOGY, Vol. 20, issued
February 1990 , Buckle et al . , "Human Memory T cells
Express Intercellular Adhesion. Molecule-1 Which Can
Be Increased by Interleukin-2 and Interferon-c* ,
pages 337-341, See Entire Document.
1-18, 52-53
19-32, 48-53
19-32, 48-53
1-17, 52-53
1-32, 48-53
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